Nucleic Acids Research, 2008, Vol. 36, No. 15Published online 25 July 2008
60S ribosomal subunit assembly dynamics
defined by semi-quantitative mass
spectrometry of purified complexes
Alice Lebreton1, Jean-Claude Rousselle2, Pascal Lenormand2,
Abdelkader Namane2, Alain Jacquier1, Micheline Fromont-Racine1
and Cosmin Saveanu1,*
1Institut Pasteur, Unite ´ de Ge ´ne ´tique des Interactions Macromole ´culaires, CNRS-URA2171 and
2Institut Pasteur, Plate-Forme Prote ´omique, 75724 Paris Cedex 15, France
Received April 30, 2008; Revised July 2, 2008; Accepted July 5, 2008
During the highly conserved process of eukaryotic
ribosome formation, RNA follows a maturation path
with well-defined, successive intermediates that
dynamically associate with many pre-ribosomal
assembly process is still lacking. To obtain data
on the timing and order of association of the differ-
ent pre-ribosomal factors, a strategy consists in the
use of pre-ribsomal particles isolated from mutants
that block ribosome formation at different steps.
Immunoblots, inherently limited to only a few fac-
tors, have been applied to evaluate the accumula-
tion or decrease of pre-ribosomal intermediates
under mutant conditions. For a global protein-level
description of different 60S ribosomal subunit
maturation intermediates in yeast, we have adapted
a method of in vivo isotopic labelling and mass
spectrometry to study pre-60S complexes isolated
from strains in which rRNA processing was affected
by individual depletion of five factors: Ebp2, Nog1,
Nsa2, Nog2 or Pop3. We obtained quantitative data
for 45 distinct pre-60S proteins and detected coor-
dinated changes for over 30 pre-60S factors in the
analysed mutants. These results led to the charac-
terisation of the composition of early, intermediate
and late pre-ribosomal complexes, specific for
crucial maturation steps during 60S assembly in
Eukaryotic ribosome biogenesis is a complex cellular
pathway, which requires a large number of proteins,
stably or transiently associated to many macromolecular
complexes. This pathway results in the production of
mature ribosomal subunits, the small one (40S), composed
in yeast of 33 ribosomal proteins (r-proteins) and assem-
bled with the 18S ribosomal RNA (rRNA) and the large
one (60S) containing 48 r-proteins and the 25S, 5.8S and
5S rRNAs. The RNA component of the ribosomes is
synthesized in the nucleolus as the 35S and 5S rRNA
precursors. A large number of proteins and snoRNA
associate co- or post-transcriptionally to the large 35S
RNA and form, in association with U3 snoRNA, large
particles known as 90S, or the SSU-processome (1,2).
Pre-60S and pre-40S particles, formed from the initial
large precursors, follow two distinct maturation pathways
in the nucleoplasm and the cytoplasm. The pre-rRNAs are
gradually modified and processed, and the subunits
achieve their structure and translating capacity.
More than 300 proteins have been characterised or pre-
dicted to assist the pre-ribosomal particles during their
maturation. Many of these factors are transiently asso-
ciated with the 90S, pre-60S and pre-40S particles and
are known as pre-ribosomal proteins. Functional studies
on pre-ribosomal factors could be undertaken once the
composition of pre-ribosomal complexes had been deter-
mined by affinity purification experiments (3–5). Such
experiments gave limited information on the positioning
of the identified proteins in the pathway. Affinity purifica-
tions of pre-ribosomal factors and their associated
Alice Lebreton, E´quipe Labellise ´ e La ligue, Centre de Ge ´ ne ´ tique Mole ´ culaire, CNRS UPR2167 associe ´ e a ` l’Universite ´ P. et M. Curie, Avenue de la
Terrasse, 91198 Gif-sur-Yvette, France
*To whom correspondence should be addressed. Tel: þ1 33 140613431; Fax: þ1 33 140613456; Email: email@example.com
? 2008 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/
by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
complexes result in the isolation of heterogeneous mix-
tures of intermediate particles because some factors
associate very transiently with the pre-ribosomal particles,
whereas others follow the process from the nucleolus to
the cytoplasm. To isolate and characterise discrete inter-
mediates in ribosome biogenesis, the purification of
enriched complexes involved in a given step was per-
formed in mutant strains (6–9). These intermediates were
also analysed by testing the presence of known pre-rRNAs
enriched in association with different pre-ribosomal or
r-proteins (2). When antibodies are available, the presence
of specific factors in the purified complexes under native
and mutant conditions can be tested. The obtained results
provide valuable, but limited information about the order
of sequential association or dissociation of a number of
factors during the assembly of 60S or 40S particles.
An alternative to the antibodies-based detection of pro-
teins in the isolated complexes is quantitative mass spec-
trometry. One of its versions, using isotopically labelled
peptides, the iTRAQ methodology (10), was used to
compare the composition of complexes associated with
Nop7-TAP under wild-type conditions and when the
GTPase Nog1 was mutated (11). Only modest differences
were observed, emphasizing the importance of the chosen
mutant for this type of analysis. However, the results
demonstrated that mass spectrometry is a suitable
method for the quantitative analysis of the composition
of pre-ribosomal complexes.
The present work aimed at establishing the assembly
order of pre-60S factors during ribosome biogenesis,
which would allow the composition of intermediate com-
plexes at given steps to be defined. To this end, we analysed
mutants that specifically accumulate different pre-rRNAs,
and thus affect the pathway at early, intermediate or late
steps, before the export of pre-60S particles to the cyto-
plasm. We compared the composition of the purified
complexes under native or mutant conditions by semi-
quantitative mass spectrometry, using a method derived
from SILAC (Stable Isotope Labelling with Amino-acids
in Cell culture) (12). The obtained results were in excellent
correlation both with previously published data on specific
factors and with the overall topology of the large-scale
network of affinity-purification interactions, validating
the approach. Moreover, we could define groups of early,
intermediate and late associating proteins that accurately
describe pre-60S assembly dynamics in yeast.
MATERIALS AND METHODS
Yeast strains used in this work are listed in Supplementary
Table S1. LMA335 (PGAL1-NOG1), LMA336 (PGAL1-
EBP2) and LMA343 (PGAL1-POP3) conditional mutant
strains were generated by homologous recombination
using PCR products to transform BY4742 strain. PCR
fragments containing the repressible GAL1 promoter
flanked by sequence upstream and downstream the ATG
of each gene respectively were synthesized from pFA6a-
kanMX6-PGal1 plasmid (13). LMA368 was generated by
mating LMA355 (MAK11-TAP) with LMA340. After
sporulation and dissection, the spores were selected for
histidine prototrophy and for the resistance to the
Geneticin. LMA406 and LMA409 strains were obtained
by transformation of LMA160 (RLP24-TAP) strain with
a PCR product containing the repressible GAL1 promoter
flanked by sequence upstream and downstream the ATG
of either NOG2 or POP3 respectively.
Interaction data collection and network visualisation
A network of physical interactions involving known or
putative pre-60S factors was built to assess visually the
distribution of proteins that were showing significant
changes in the purified complexes under mutant condi-
tions. The factors that were either annotated as involved
in ribosome biogenesis (SGD database) or were linked
to pre-ribosomal factors by showing strong mRNA
co-variation under various environmental conditions
were selected. Co-purification data involving the selected
323 proteins were recovered from a total of 28 462 inter-
actions available in the BioGrid database (14), correspond-
ing to 32 individual publications, including large-scale
TAP analyses (15–18). A network of interactions was
obtained using factors showing at least 3 known interac-
tions with other proteins involved in ribosome biogenesis
and a sub-network, corresponding to pre-60S proteins was
selected in Cytoscape (19). An automatic graph layout
algorithm was used to distribute the proteins (nodes in
the network) accordingly to their interactions (edges).
Colours were assigned to nodes, depending on the
observed variations in the SILAC experiments using a
threshold of 2 to depict significant changes in protein
levels under mutant conditions.
RNA extraction, primer extension and northern blot
After a preculture in rich medium containing galactose,
the cells were incubated in rich medium containing glucose
for various times from 0 to 8 or 28h. Total RNAs were
extracted using standard glass beads and phenol proce-
dure. Primer extension was done with32P-labelled oligo-
nucleotides CS10 (CGC CTA GAC GCT CTC TTC TTA)
and MFR457 (GCT TAA AAA GTC TCT TCC CGT CC)
specific of U2 snRNA used as an internal control; the
products were separated on 5% polyacrylamide-urea gels.
For northern blots, after separation of RNA on 5%
polyacrylamide-urea gels and transfer on Hybond-Nþ
membranes, 7S, 5.8S and U6 snRNA were probed with
32P-labelled oligonucleotides CS3 (GGC CAG CAA TTT
CAA GTT A), CS5 (CGG AAT TCT GCA ATT CAC
ATT ACG) and MFR555 (AAC TGC TGA TCA TCT
CTG) respectively. The position of pre-ribosomal probes
is illustrated on Figure 1A.
Purification of complexes forSILAC quantification
We adapted an isotopic labelling method (SILAC) based
on mass spectrometry to distinguish quantitative changes
in complexes composition under different conditions (12).
For each experiment, we followed the protocol outlined
in Figure 2A. We cultivated either the wild type or the
mutant strains in deuterated leucine, glucose-containing
Nucleic Acids Research, 2008, Vol. 36, No. 154989
minimal medium for at least 6 generations, so that all
leucines were deuterated. The other strain was cultivated
in normal leucine, glucose-containing minimal medium.
The period of time for the shift to glucose was determined
for each mutant according to its growth curve (Supple-
mentary Figure S1). LMA182 (PGAL1-NOG1) was grown
for 16h in glucose-containing medium, LMA406 (PGAL1-
NOG2) for 17h, LMA272 (PGAL1-NSA2) for 16h,
LMA368 (PGAL1-EBP2) for 15h, and finally LMA409
(PGAL1-POP3) for 15h. The 600nm optical densities of
the recovered cultures were between 1 and 2.
The complex purifications were performed as described
(20), starting from 4l of yeast cultures and using buffers
containing 0.1M NaCl. The same amount of purified
complex, as estimated by the Bradford method or by
Coomassie blue staining on analytical polyacrylamide
Figure 1. (A) Schematic representation of the rRNA maturation pathway. The oligonucleotides used in this study are indicated. (B) Total RNAs
were extracted from the different strains after growth in galactose-containing, synthetic complete medium or after shift to glucose medium for either
8 or 28h. 27SA2and 27SB rRNA intermediates were detected by primer extension using oligonucleotide CS10, on a 5% polyacrylamide-urea gel.
The U2 snRNA was used as a loading control. (C) 7S intermediate and 5.8S mature rRNA were separated on 5% polyacrylamide-urea gel and
detected by northern blot using32P-labelled oligonucleotides CS3 or CS5. The U6 snRNA was used as a loading control.
Nucleic Acids Research, 2008, Vol. 36, No. 15
gels, from either the wild type or the mutant strains were
mixed and separated on a 5–20% polyacrylamide gradi-
ent-SDS gel. After electrophoresis, the proteins were
stained with colloidal Coomassie blue, and then identified
and quantified by mass spectrometry.
All the results were obtained on a MALDI-TOF
Voyager DE-STR mass spectrometer (Applied Biosys-
tems). Protein identifications were obtained with MS-Fit
(Baker, P.R. and Clauser, K.R. http://prospector.ucsf.
edu, version 3.1.1) using a yeast proteome database
to which common keratin contaminants and trypsin
sequences had been added. All the calibrated spectra
were exported to text files for quantitative analysis. For
each spectrum, the peaks that corresponded to an identi-
fied protein were assigned to the corresponding peptide
sequences. The peaks corresponding to leucine-containing
peptides were manually analysed and the ratio between the
deuterated and non-deuterated monoisotopic peak heights
(with background subtracted) were further used in the
analysis. Python scripts were used to extract relevant spec-
tra region on the basis of MS-Fit proteins identifications,
convert these spectra to a custom XML format and inter-
actively validate the results. A summary of the quantifica-
tion results is available in Supplementary Table S2, and
more details about the number of quantified peptides in
Supplementary Table S3.
Proteins were separated on a 10% polyacrylamide-SDS
gel and transferred on a nitrocellulose membrane. Specific
proteins were detected by indirect immunoblot, using spe-
cific polyclonal rabbit antibodies at a 1:5000 dilution.
Secondary antibodies (Goat Anti-Rabbit-HRP Conjugate
from Bio-Rad) were used at a 1:10000 dilution.
Choosing mutants and tagged pre-60S factorsto analyse
To obtain an overview of the composition of the succes-
sive 60S precursor complexes, we defined a set of 83
proteins that are known or predicted to be involved in
60S formation and collected their physical interaction
results (co-purification experiments) from the BioGrid
database (14). Taken individually, each pre-60S factor
that was used as a bait, co-purified with a number of
other proteins, providing a profile of the factors that can
Protein identification by MALDI-TOF
Peptide signal quantification
peptide from W (2 Leu)
1:1 mix; SDS-PAGE
Complete gel slicing
peptide from Z (2 Leu)
Deuterated percent (input)
40 60 80100
Deuterated percent (measured)
Figure 2. Adaptation of SILAC to the quantitative analysis of pre-
ribosomal complexes. (A) Schematics of the TAP-SILAC coupling for
the quantitative analysis of protein composition changes. Two strains
producing a TAP-tagged bait protein ‘X’, a wild type and a mutant
expressing a glucose-repressible pre-ribosomal gene ‘Y’ are grown in
glucose-containing minimal medium, either in presence of normal
leucine, or in presence of deuterated leucine. X-TAP-associated com-
plexes are purified by tandem affinity purification, then the wild type
and mutant samples are mixed together and separated by SDS-PAGE.
Specific protein bands are cut out from the gels and analysed by
MALDI-TOF spectrometry. (B) The non-deuterated/deuterated ratio
can be quantified for each identified peptide in a given protein, using
the mass spectra. As an example, signals obtained with a peptide from
protein ‘Z’, which accumulates in the mutant and a peptide from pro-
tein ‘W’, which is lost in complexes purified from the mutant strain
were illustrated. (C) To test the performance of mass spectrometry
quantification, total yeast protein extracts from cells grown in
medium containing leucine or deuterated leucine were mixed in differ-
ent ratios, with a percentage of deuterated proteins from 0% to 100%.
After separation by SDS-PAGE, four bands at different molecular
weights (range from 12–125kDa) were recovered and proteins identified
by MALDI-TOF mass spectrometry. Several peptide signals were
quantified in the different samples (8–14 values, depending on the spec-
tra quality) and a box and whiskers plot for the measured values were
represented in correlation with the expected percentage of deuterated
leucine-containing peptides. Boxes represent the upper and lower quar-
tile, the horizontal line, the median and the bars represent the maxi-
mum and minimum measured values.
Nucleic Acids Research, 2008, Vol. 36, No. 154991
be concomitantly found on pre-60S particles. To assign
these factors to groups, we used the data to build a
matrix of physical interactions, constituted of 49 tagged
proteins, in rows, and 63 distinct associated proteins,
in columns (Supplementary Figure S2). We expected
proteins involved in the same pre-60S precursors to be
grouped together and, as a result, to gain insights into
the timing of association and dissociation of different
pre-60S factors. Even though this kind of analysis
proved previously to be efficient to distinguish 90S, pre-
60S and pre-40S particles when using a large number of
pre-ribosomal proteins and their associations (3), the
obtained cluster of pre-60S factors was unable to further
refine the involvement of many of the proteins at specific
steps in the pathway.
To get a detailed description of the composition of pre-
60S complexes along the pathway, other methods had
to be set up. One such method uses quantitative mass-
spectrometry to compare the composition of purified
pre-ribosomal particles from mutant strains affected for
60S biogenesis. For this type of study, both the choice
of mutants and tagged proteins used for the purification
of the complexes were important. We chose 5 pre-60S
factors (Ebp2, Nog1, Nsa2, Pop3 and Nog2) to block
the biogenesis of the large ribosomal subunit at specific
steps, defined by the accumulation of distinct pre-rRNA
intermediates (6,21–24). As these factors are essential for
cell viability, we generated strains where the genes encod-
ing these factors were under the control of a glucose-
repressed promoter (GAL1-10). All these strains showed
a progressive growth defect when cultivated in glucose-
containing medium (Supplementary Figure S1). The
growth defect was associated with changes in the levels
of different rRNA maturation intermediates (Figure 1A),
tested by primer extensions (Figure 1B) and northern blot-
ting (Figure 1C). As expected from previous reports on
the role of these factors, we observed different effects of
the proteins depletion on the steady-state levels of the pre-
60S RNA components. Like the ebp2-1 mutant (21), the
repression of EBP2 led to early defects in the 60S matura-
tion pathway, with a 27SA2 increase and a decrease
in 27SB and 7S pre-rRNA levels. Repression of POP3
expression induced changes in the 27SBL/27SBS, 7SL/7SS
and 5.8SL/5.8SSratios, which have been previously attrib-
uted to the role of the RNase MRP (Pop3 being one of its
subunits) in the RNA cleavage at site A3(22). As pre-
viously described (6,23), the repression of either NOG1
or NSA2 expression affected the cleavage of 27SB precur-
sors at site C2, hence preventing the formation of 7S.
Nog2 depletion had a different effect on 60S biogenesis
at a late nuclear step with the accumulation of both
27SB and 7S species, as reported (24). Thus, using these
five mutants, the 60S biogenesis could be blocked at early,
intermediate or late nuclear steps.
We combined the glucose-repressible strains with geno-
mic TAP-tagging of Rlp24, a factor that associates with
pre-60S particles both in the nucleus and the cytoplasm
(6), allowing the isolation of blocked complexes in the
different mutants. We still needed a robust method for
quantitative comparisons of the protein composition of
these isolated complexes.
Adaptation ofSILAC to theanalysis of TAP complexes
Several methods of quantitative mass spectrometry, based
on isotopic labelling of proteins or peptides have been
described (25,26). To monitor the relative amounts of
each purified protein in wild type and mutant samples,
we chose SILAC, as a robust method of in vivo labelling
that could be adapted to the quantification of simple MS
spectra obtained with a MALDI-TOF mass spectrometer.
SILAC is based on the differential labelling of two cul-
tures before the analysis, for example, a wild type and a
mutant strain. Proteins from one of the samples incorpo-
rate a deuterated amino acid, leucine with 3 deuterium
atoms in our case, which leads to a shift in the m/z for
all the peptides coming from that sample, when compared
with a sample coming from a culture grown with non-
deuterated leucine (12). For each single-charged peptide
containing a leucine, this results in a shift of 3 Da in the
mass spectrum. Assignment of the peptide peaks to identi-
fied proteins and measurement of the pairs (deuterated/
non-deuterated) peak heights in the MALDI-TOF spectra
provide a relative quantitative measure for the initial con-
tent of the protein in the two samples. To limit the analysis
to the changes in composition of specific complexes, an
additional TAP purification step was required; the whole
procedure is illustrated in Figure 2A. The results obtained
for different proteins can be explained by following the
example shown in Figure 2B. Proteins that are no longer
co-purified with the tagged protein in the mutant strain
generate spectra profiles resembling that shown for the
protein marked ‘W’. In contrast, proteins that are
enriched in the mutant complexes result in peptide profiles
like the one shown as example ‘Z’. Both shown peptides
have a single charge and contain 2 leucines, resulting in a
6 Da shift in the detected m/z.
Since our method only relies on the identification of
proteins by their peptide mass fingerprint, we tested the
reproducibility and precision of quantitative measures
that are based on MALDI-TOF MS spectra. We mixed
in different proportions total protein extracts from strains
grown with deuterated or non-deuterated leucine and
obtained an excellent correlation between our measures
Defining theassociation of proteins toearly
or late 60Sprecursors
The depletion of many pre-60S factors results in a relative
27SB pre-rRNA accumulation (3,5). To assign these
different proteins to groups that would predict their
early or late association to the ribosome precursors, we
first tested the effects of Nog1 depletion on the composi-
tion of Rlp24-TAP associated complexes. Our previous
data, based on immunoblots, indicated that under these
conditions several important changes in the composition
of the purified complexes occur, like the accumulation of
Mak11 or the depletion of Nog2 (6). Characterisation
of Mak11 and Nog2 confirmed their involvement in
early or late steps of nuclear 60S maturation, respectively
(24,27). We thus began our investigation of the order of
assembly of 60S ribosomal subunits with an analysis of the
Nucleic Acids Research, 2008, Vol. 36, No. 15
changes in the composition of Rlp24-TAP-associated
complexes when Nog1 was depleted.
In addition to large subunit r-proteins and usual con-
taminants (Ssb, translation elongation factors or small
subunit r-proteins), we identified and quantified the rela-
tive abundance of 28 pre-60S factors in the purified com-
plexes (Figure 3A and B). As expected, one of the proteins
showing the largest decrease in the particles isolated from
the mutant strain, was the depleted protein Nog1. Other
factors, like Nog2 or Nug1 showed a similar behaviour,
in good correlation with previous descriptions of the late
association of these factors to pre-60S complexes (24,28).
Conversely, proteins like Ssf1, described as acting early in
the 60S biogenesis (29), were enriched in the complexes
isolated under mutant conditions.
Many quantified pre-60S factors (located in the grey
area on Figure 3A and B) were decreased when compared
to the levels of Rlp24-TAP, the protein used for the iso-
lation of the particles, but were enriched in the complexes,
when compared to the average level of the quantified ribo-
somal proteins (indicated by dashed lines in Figure 3A).
In the absence of an absolute standard, we arbitrarily fixed
the measured ratio for Rlp24-TAP to 1 and reported the
results for the other proteins accordingly. If we assume
that the ribosomal proteins decreased under mutant
conditions in the complexes isolated in association with
Rlp24-TAP, a straightforward explanation would be
that a fraction of the Rlp24-TAP bait was no longer asso-
ciated with large complexes when ribosome biogenesis was
blocked. While changing the reference has an impact on
Figure 3. Rlp24-TAP combined with Nog1 depletion defines classes of pre-60S factors. (A) Scatter plot of the mutant/wild-type ratio obtained for
each protein in two independent experiments where, in turn, the wild type or the mutant strain were cultivated in deuterated leucine-containing
medium. Bait (Rlp24) is indicated by a filled square; Proteins enriched in the complexes purified from the mutant strain when using the bait protein
as reference are indicated as upward pointing filled triangles; Proteins enriched or in similar amounts in the wild type and mutant complexes when
using ribosomal proteins (Rpl) as reference are indicated as empty triangles; Proteins showing decreased levels in the complexes purified from the
mutant strain (downward pointing filled triangles); Large ribosomal subunit proteins (filled circles); Contaminants (empty circles). Dashed lines
indicate the average ratios obtained for large subunit r-proteins. (B) Quantifications of the mutant/wild-type ratios for proteins identified in Rlp24-
TAP-associated complexes purified from strains expressing NOG1 or not. Boxes represent the average value of the ratio for all quantified peptides in
a given protein in two distinct experiments. Error bars indicate 95% confidence intervals. (C) Immunoblot validation of the SILAC quantifications.
Aliquots from Rlp24-TAP-associated complexes recovered from the wild type or the PGAL1-NOG1 strain were separated by SDS-PAGE and the
amounts of Rlp24, Mak11, Nop7, Nog1, Nog2 or Arx1 were assessed by western blot using specific antibodies. Both the eluate from the first
purification step on IgG-sepharose (TEV) and the final eluate (TAP) were analysed (left and right panels, respectively).
Nucleic Acids Research, 2008, Vol. 36, No. 154993
what can be considered as an increase or a decrease in
protein amounts, it does not modify the classification of
the ratios for the different pre-60S factors.
To validate the results of mass spectrometry quantifica-
tions we performed immunoblots on TEV and TAP
purifications, using antibodies that recognise specifically
Rlp24, Mak11, Nop7, Nog1, Nog2 or Arx1 (Figure 3C).
The results were in good agreement with the mass-
spectrometry-based measurements, hence validating the
To confirm and reinforce the data from our first experi-
ments, we used another mutant having a similar pre-
rRNA phenotype and another protein as bait. We chose
Nsa2 depletion as mutant condition since it led to the
same pre-rRNA species accumulation as the depletion of
Nog1 (Figure 1B). In addition, Nsa2 was the protein
showing the strongest decrease in the Rlp24-TAP com-
plexes when Nog1 was depleted (Figure 3A and B), an
effect explained by the observation of Nsa2 destabilisation
under these conditions (23). Nog1-associated proteins are
very similar to those found in association with Rlp24 (6).
We obtained quantitative data, by TAP-SILAC for Nog1-
TAP under wild type or Nsa2 depletion conditions for 30
pre-60S factors (Figure 4A and B). Twenty-three proteins
were quantified in both Rlp24-TAP, Nog1 depletion and
Nog1-TAP, Nsa2 depletion experiments (Supplementary
Figure S3), with a very good Kendall tau rank correla-
tion coefficient (t¼0.727) of the observed changes
(p¼1.5?10?6that the values are not correlated). The
results obtained in two independent experiments with dif-
ferent mutants and different TAP-tagged proteins allowed
the definition of two groups of pre-60S factors. An early
one is composed by proteins that are likely to be present
on pre-60S particles before the step blocked in the absence
of either Nog1 or Nsa2, and is composed of at least 20
different proteins, including factors like Ssf1 or Mak11.
Proteins that showed reproducibly diminished levels in
the complexes under mutant conditions (Nop53, Nog2,
Nug1 or Rsa4) are likely to associate to the 60S precursors
at a later stage. The different abundance of proteins in the
early and late group of pre-60S factors is correlated with
the progressive simplification of pre-60S particles during
their maturation (30).
The early pre-60S factorscan be separatedin subgroups
In view of the successful SILAC-based measurements of
pre-60S factors redistribution under Nog1 and Nsa2
depletion, we sought to refine the results by testing the
effects of depleting an earlier acting factor (as defined by
pre-rRNA accumulation studies), Ebp2. No protein could
be observed accumulating in association with Rlp24 under
Ebp2 mutant conditions, when compared with the wild
type (Supplementary Figure S4A and B). These results
suggest that Ebp2 action precedes the association of
Rlp24 to the pre-60S particles and that an earlier protein
should be used to isolate the complexes that accumulate
in the absence of Ebp2. As we have previously shown
that Mak11 might already be present on the complexes
at a moment preceding Rlp24 association to pre-60S
particles (27), we tested the influence of Ebp2 depletion
A number of proteins, and especially Noc2, Nop4 and
Rrp5, where present in higher amounts in the Mak11-
associated complexes in the mutant (Figure 5A and B),
Average relative ratio
ratio mutant/wt (exp. 1)
ratio mutant/wt (exp. 2)
Figure 4. Nog1-TAP combined with the depletion of Nsa2 defines
C2-specific 60S precursors. (A, B) Legends are as for Figure 3, except
that the bait was Nog1, and the depleted protein, Nsa2.
Nucleic Acids Research, 2008, Vol. 36, No. 15
in good agreement with the function of these proteins
early in the pathway (1,31,32). On the contrary, some of
the proteins that were classified as ‘early’ in respect with
Nog1 or Nsa2 function were lost when Ebp2 was depleted,
for example Nop16, Erb1 or Nop7.
We conclude that Ebp2 is required for the progression
of the 60S assembly pathway at an early stage and affects
the specific association of a large number of pre-60S
factors to the nascent particles.
Nog2isrequired forthe association of late pre-60S factors
Since we were able to define subgroups of pre-60S
factors that associate early in the pathway, we wondered
if we could distinguish different classes of late, nuclear-
associating pre-60S proteins. Among the different proteins
that we defined as late-associating, we chose Nog2 deple-
tion as the mutant condition coupled with the isolation of
pre-60S complexes in association with Rlp24-TAP for
further experiments. Unlike Arx1 or Tif6, Nog2 is strictly
nuclear and its depletion leads to the accumulation of
the last nuclear pre-rRNA intermediate, 7S (Figure 1C)
and also blocks the export of the 60S precursors to the
Little change was observed in Rlp24-TAP-associated
complexes between the wild-type conditions and the deple-
tion of Nog2 (Figure 6A and B). The fact that both 7S and
27S species are accumulated in Nog2-depleted cells might
provide an explanation for the lack of amplitude of the
observed effects. Still, Rsa4 and Arx1 levels decreased in
these complexes, suggesting that their association to late
pre-60S depends on Nog2 presence. Rsa4 was previously
described as a factor required for late pre-60S biogenesis
(33), but its precise binding step had not been specifically
addressed. Recently, Arx1 was described as a nuclear
export receptor for the 60S precursors (34,35); its decrease
in the Nog2 mutant is thus in good agreement with a late
role of Arx1 in 60S biogenesis. Changes in the composi-
tion of 60S particles, detected by quantitative mass spec-
trometry, can be thus useful to orient further functional
characterisation of specific pre-60S factors.
The twodistinct routes of 27SBformationinvolve
asimilar set ofpre-60S factors
Our experiments were based on the hypothesis that pro-
teins association to the different intermediates follows,
as the RNA maturation, a linear order, with every pre-
60S particle deriving from a preceding one. We wondered
whether protein composition may have an influence on the
pre-rRNA processing and if particles of similar pre-rRNA
components may contain different protein sets. To this
end, we explored the effects of an RNase MRP mutant
on Rlp24-TAP-associated complexes composition. After
A2cleavage that leads to the 27SA2precursor, processing
of 27S species can follow two distinct routes (Figure 1A):
the major one involves a cleavage at site A3by RNase
MRP; the minor one consists of a 50-30exonucleolytic
trimming by a yet unidentified ribonuclease, and leads to
the production of 50-extended species (27SBL, 7SLprecur-
sors and 5.8SLrRNA). The repression of POP3 impairs
RNase MRP function, leading to a complete redistribu-
tion of the processing towards the minor pathway. The
analysis of Rlp24-TAP complexes in the presence or
absence of Pop3 revealed hardly any change in protein
composition (Supplementary Figure S4C and D), despite
a clear change in the ratio of the large and short form of
Average relative ratio
0.01 0.11 10100
ratio mutant/wt (exp. 2)
1/32 1/16 1/8 1/41/21248 16
ratio mutant/wt (exp. 1)
Figure 5. Early 60S precursors, isolated in association with Mak11-TAP
are enriched after Ebp2 depletion. (A, B) Legends are as for Figure 3,
except that the bait was Mak11, and the depleted protein, Ebp2.
Nucleic Acids Research, 2008, Vol. 36, No. 154995
the 5.8S rRNA levels (Supplementary Figure S5), suggest-
ing that except for 27SA3 processing, both the major
and the minor processing pathways involve the same set
of pre-60S factors.
Ribosome biogenesis in eukaryotes involves a large
number of factors, many of them identified during
recent large-scale co-purification studies (16–18). While
these results were crucial to define the number of different
proteins involved in this conserved pathway, they are not
suitable for a detailed characterisation of the different
intermediate ribosome precursors. Novel approaches
are needed to address this question and one of them is
the study of changes in the protein composition of
(2,6,8,9,23,36,37). We show here that coupling TAP pur-
ifications with carefully chosen mutants and novel
methods of quantitative mass-spectrometry like SILAC
(12) allows the description of groups of pre-ribosomal
factors that are likely to be functionally related. The defi-
nition of such groups may lead to oriented experiments
that would help the comprehension of the involved molec-
We integrated our results about the assembly of
pre-ribosomal factors on pre-60S particles in a simple
schematic (Figure 7A). Several groups of pre-60S factors
could be defined, on the basis of the three analysed mutant
effects. The earliest group of pre-60S factors is defined by
the proteins that accumulate during Ebp2 depletion. Since
this mutant accumulates 27SA2pre-rRNA, the first form
of RNA that is specific to the 60S assembly pathway and
signals the separation from pre-40S particles, proteins of
this group are likely to be already present on the earliest
nascent 60S precursors.
One of the most interesting findings of this study was
that some pre-60S factors, like Nop16, decreased in the
purified complexes when Ebp2 was depleted while they
accumulated under Nog1 or Nsa2 depletion, whatever
the protein used for the precursors purification. The sim-
plest explanation for this behaviour is that proteins from
this intermediate group associate to the 60S precursors at
a step that precedes the one blocked by the absence of
Nog1 or Nsa2, and follows the one blocked by Ebp2
absence (Figure 7A). The proteins that, like Mrt4, associ-
ate to the nuclear pre-60S particles at late steps depending
on the action of Nog1 and Nsa2 can be further classified in
two sub-groups, including in the latest one the factors that
also depend on Nog2 for association to the pre-60S par-
ticles (for instance, Rsa4).
Our data cannot indicate with precision the moment of
dissociation for most of the identified proteins because the
lack of identification of a protein in a given complex is not
an absolute proof for its absence from that complex.
However, when a protein could be identified under some
conditions in a complex but was absent from similar com-
plexes purified with another bait protein, such information
was pragmatically used to differentiate subgroups of pre-
60S factors. With the above limitations in mind, we dis-
tinguished the proteins that looked specific to the early
Mak11-associated complexes (for instance Dbp7) from
those that were also present on later Rlp24 or Nog1-asso-
ciated pre-60S particles (for instance Mak5). Except for
known shuttling factors that are exported to the cyto-
plasm, the estimation of the point where other factors
ratio mutant/wt (exp. 2)
ratio mutant/wt (exp. 1)
1/32 1/161/8 1/41/212
Average relative ratio
0.01 0.11 10100
Figure 6. Modest changes in the composition of late nuclear pre-60S
complexes are induced by Nog2 depletion. (A, B) Legends are as for
Figure 3, with Rlp24 as the tagged protein and Nog2 as the depleted
Nucleic Acids Research, 2008, Vol. 36, No. 15
Figure 7. SILAC-based definition of pre-60S sub-complexes. (A) Based on the results of our analysis we deduced possible association and
dissociation steps that define several groups of pre-60S factors, relative to the involvement of Ebp2, Nog1/Nsa2 and Nog2 in the rRNA maturation
pathway. (B, C, D) The results of the SILAC quantifications were colour-coded and superposed on a Cytoscape representation of pre-60S proteins,
showing identified bait–prey interactions from published affinity purification experiments. We first extracted a 60S-specific sub-network from the
larger pre-ribosomal interactions network. The network layout was obtained by applying the yFiles organic algorithm in Cytoscape. The represented
experiments are (B) Mak11-TAP, depletion of Ebp2, (C) Rlp24-TAP, depletion of Nog1 and (D) Nog1-TAP, depletion of Nsa2. The bait is framed
by a blue line, and the depleted factor in a white disk dashed in red. Factors which were enriched in complexes isolated from the mutant strain at
least by a factor of 2 are shown in orange, factors which were decreased by >50% are in cyan, factors of unchanged levels are in dark grey disks.
Light grey disks indicate factors that we identified in at least one of our experiments. Light yellow disks correspond to known pre-60S factors which
were not identified in the SILAC experiments.
Nucleic Acids Research, 2008, Vol. 36, No. 154997
leave the particles would need further validation by
Our analysis provides new insights into the assembly
scenario for the eukaryotic large ribosomal subunit in
yeast, by describing the protein composition of distinct
assembly intermediates along the pathway. The results
were in excellent correlation with the topology of the net-
work of physical association of pre-60S factors. Proteins
that were enriched or decreased in precursor particles
under mutant conditions were clustered in specific regions
of the graph, depending on the analysed protein depletion
(Figure 7B, C and D). Our results are also in agreement
with previously published data about the pre-60S factors
function or association to the pre-ribosomal complexes.
For instance, Dbp7, Dbp9, Nop4, Noc2, Rpf2, Ebp2,
Rrp1 and Ssf1 were known for their participation in
early steps of the large ribosomal subunit biogenesis
(8,21,29,31,32,38–42), and Rrp5 is a component of the
SSU-processome (1). Nug1 was characterised for its func-
tions in the late steps of biogenesis, at the same time as
Nog2 (28) and Arx1 are both involved in the export of
pre-60S particles from the nucleus (34,35).
On the basis of the analysis of three distinct 60S biogen-
esis steps (depending on Ebp2, Nog1/Nsa2 and Nog2) we
were thus able to obtain an unbiased panorama of the
association timing for 30 pre-60S factors during ribosome
biogenesis. These results, integrated into the physical asso-
ciation map for all the previously identified pre-60S
proteins, and combined with data about the molecular
function of each pre-60S factor in the biogenesis, are
useful to build a view of protein dynamics in the ribosome
Supplementary Data are available at NAR Online.
We are grateful to Franc ¸ oise Stutz (Centre Me ´ dical
Universitaire, Gene ` ve, Switzerland) for the pFA6a-TAP-
Tag-His3MX6 vector. We thank Yanhua Yao and
Tommaso Villa for critical reading of the manuscript.
This work was supported by the Ministe ` re de ´ le ´ gue ´ a `
l’Enseignement Supe ´ rieur et a `
BCM0089-2003). Funding to pay the Open Access publi-
cation charges for this article was provided by the
Ministere delegue a l’enseignement superieur et a la
recherche, France in the form of the ACI-BCM0089-
la Recherche (ACI-
Conflict of interest statement. None declared.
1. Dragon,F., Gallagher,J.E., Compagnone-Post,P.A., Mitchell,B.M.,
Porwancher,K.A., Wehner,K.A., Wormsley,S., Settlage,R.E.,
Shabanowitz,J., Osheim,Y. et al. (2002) A large nucleolar U3
ribonucleoprotein required for 18S ribosomal RNA biogenesis.
Nature, 417, 967–970.
2. Ferreira-Cerca,S., Poll,G., Kuhn,H., Neueder,A., Jakob,S.,
Tschochner,H. and Milkereit,P. (2007) Analysis of the in vivo
assembly pathway of eukaryotic 40S ribosomal proteins. Mol. Cell,
3. Fromont-Racine,M., Senger,B., Saveanu,C. and Fasiolo,F. (2003)
Ribosome assembly in eukaryotes. Gene, 313, 17–42.
4. Tschochner,H. and Hurt,E. (2003) Pre-ribosomes on the road from
the nucleolus to the cytoplasm. Trends Cell Biol., 13, 255–263.
5. Henras,A.K., Soudet,J., Gerus,M., Lebaron,S., Caizergues-
Ferrer,M., Mougin,A. and Henry,Y. (2008) The post-transcriptional
steps of eukaryotic ribosome biogenesis. Cell Mol. Life Sci.,
6. Saveanu,C., Namane,A., Gleizes,P.E., Lebreton,A., Rousselle,J.C.,
Noaillac-Depeyre,J., Gas,N., Jacquier,A. and Fromont-Racine,M.
(2003) Sequential protein association with nascent 60S ribosomal
particles. Mol. Cell Biol., 23, 4449–4460.
7. Miles,T.D., Jakovljevic,J., Horsey,E.W., Harnpicharnchai,P.,
Tang,L. and Woolford,J.L. Jr. (2005) Ytm1, Nop7, and Erb1 form
a complex necessary for maturation of yeast 66S preribosomes.
Mol. Cell. Biol., 25, 10419–10432.
8. Zhang,J., Harnpicharnchai,P., Jakovljevic,J., Tang,L., Guo,Y.,
Oeffinger,M., Rout,M.P., Hiley,S.L., Hughes,T. and
Woolford,J.L. Jr. (2007) Assembly factors Rpf2 and Rrs1
recruit 5S rRNA and ribosomal proteins rpL5 and rpL11 into
nascent ribosomes. Genes Dev., 21, 2580–2592.
9. Perez-Fernandez,J., Roman,A., De Las Rivas,J., Bustelo,X.R. and
Dosil,M. (2007) The 90S preribosome is a multimodular structure
that is assembled through a hierarchical mechanism. Mol. Cell Biol.,
10. Ross,P.L., Huang,Y.N., Marchese,J.N., Williamson,B., Parker,K.,
Hattan,S., Khainovski,N., Pillai,S., Dey,S., Daniels,S. et al. (2004)
Multiplexed protein quantitation in Saccharomyces cerevisiae using
amine-reactive isobaric tagging reagents. Mol. Cell Proteomics, 3,
11. Fuentes,J.L., Datta,K., Sullivan,S.M., Walker,A. and
Maddock,J.R. (2007) In vivo functional characterization of
the Saccharomyces cerevisiae 60S biogenesis GTPase Nog1.
Mol. Genet. Genomics, 278, 105–123.
12. Ong,S.E., Blagoev,B., Kratchmarova,I., Kristensen,D.B., Steen,H.,
Pandey,A. and Mann,M. (2002) Stable isotope labeling by amino
acids in cell culture, SILAC, as a simple and accurate approach to
expression proteomics. Mol. Cell Proteomics, 1, 376–386.
13. Longtine,M.S., McKenzie,A. III, Demarini,D.J., Shah,N.G.,
Wach,A., Brachat,A., Philippsen,P. and Pringle,J.R. (1998)
Additional modules for versatile and economical PCR-based gene
deletion and modification in Saccharomyces cerevisiae. Yeast, 14,
14. Breitkreutz,B.J., Stark,C., Reguly,T., Boucher,L., Breitkreutz,A.,
Livstone,M., Oughtred,R., Lackner,D.H., Bahler,J., Wood,V. et al.
(2008) The BioGRID Interaction Database: 2008 update.
Nucleic Acids Res., 36, D637–D640.
15. Krogan,N.J., Peng,W.T., Cagney,G., Robinson,M.D., Haw,R.,
Zhong,G., Guo,X., Zhang,X., Canadien,V., Richards,D.P. et al.
(2004) High-definition macromolecular composition of yeast
RNA-processing complexes. Mol. Cell, 13, 225–239.
16. Krogan,N.J., Cagney,G., Yu,H., Zhong,G., Guo,X.,
Ignatchenko,A., Li,J., Pu,S., Datta,N., Tikuisis,A.P. et al. (2006)
Global landscape of protein complexes in the yeast Saccharomyces
cerevisiae. Nature, 440, 637–643.
17. Gavin,A.C., Bosche,M., Krause,R., Grandi,P., Marzioch,M.,
Bauer,A., Schultz,J., Rick,J.M., Michon,A.M., Cruciat,C.M. et al.
(2002) Functional organization of the yeast proteome by systematic
analysis of protein complexes. Nature, 415, 141–147.
18. Gavin,A.C., Aloy,P., Grandi,P., Krause,R., Boesche,M.,
Marzioch,M., Rau,C., Jensen,L.J., Bastuck,S., Dumpelfeld,B. et al.
(2006) Proteome survey reveals modularity of the yeast cell
machinery. Nature, 440, 631–636.
19. Shannon,P., Markiel,A., Ozier,O., Baliga,N.S., Wang,J.T.,
Ramage,D., Amin,N., Schwikowski,B. and Ideker,T. (2003)
Cytoscape: a software environment for integrated models of bio-
molecular interaction networks. Genome Res., 13, 2498–2504.
20. Rigaut,G., Shevchenko,A., Rutz,B., Wilm,M., Mann,M. and
Seraphin,B. (1999) A generic protein purification method for
protein complex characterization and proteome exploration.
Nat. Biotechnol., 17, 1030–1032.
Nucleic Acids Research, 2008, Vol. 36, No. 15
21. Huber,M.D., Dworet,J.H., Shire,K., Frappier,L. and Download full-text
McAlear,M.A. (2000) The budding yeast homolog of the human
EBNA1-binding protein 2 (Ebp2p) is an essential nucleolar protein
required for pre-rRNA processing. J. Biol. Chem., 275,
22. Dichtl,B. and Tollervey,D. (1997) Pop3p is essential for the activity
of the RNase MRP and RNase P ribonucleoproteins in vivo.
EMBO J., 16, 417–429.
23. Lebreton,A., Saveanu,C., Jacquier,A. and Fromont-Racine,M.
(2006) Nsa2 is an unstable, conserved factor required for
the maturation of 27 SB pre-rRNAs. J. Biol. Chem., 281,
24. Saveanu,C., Bienvenu,D., Namane,A., Gleizes,P.E., Gas,N.,
Jacquier,A. and Fromont-Racine,M. (2001) Nog2p, a putative
GTPase associated with pre-60S subunits and required for late 60S
maturation steps. EMBO J., 20, 6475–6484.
25. Ong,S.E. and Mann,M. (2005) Mass spectrometry-based proteomics
turns quantitative. Nat. Chem. Biol., 1, 252–262.
26. Mueller,L.N., Brusniak,M.Y., Mani,D.R. and Aebersold,R. (2008)
An assessment of software solutions for the analysis of mass spec-
trometry based quantitative proteomics data. J. Proteome Res., 7,
27. Saveanu,C., Rousselle,J.C., Lenormand,P., Namane,A., Jacquier,A.
and Fromont-Racine,M. (2007) The p21-activated protein kinase
inhibitor Skb15 and its budding yeast homologue are 60S ribosome
assembly factors. Mol. Cell Biol., 27, 2897–2909.
28. Bassler,J., Grandi,P., Gadal,O., Lessmann,T., Petfalski,E.,
Tollervey,D., Lechner,J. and Hurt,E. (2001) Identification of a 60S
preribosomal particle that is closely linked to nuclear export.
Mol. Cell, 8, 517–529.
29. Fatica,A., Cronshaw,A.D., Dlakic,M. and Tollervey,D. (2002)
Ssf1p prevents premature processing of an early pre-60S ribosomal
particle. Mol. Cell, 9, 341–351.
30. Nissan,T.A., Bassler,J., Petfalski,E., Tollervey,D. and Hurt,E.
(2002) 60S pre-ribosome formation viewed from assembly in the
nucleolus until export to the cytoplasm. EMBO J., 21, 5539–5547.
31. Sun,C. and Woolford,J.L. Jr. (1994) The yeast NOP4 gene product
is an essential nucleolar protein required for pre-rRNA processing
and accumulation of 60S ribosomal subunits. EMBO J., 13,
32. Milkereit,P., Gadal,O., Podtelejnikov,A., Trumtel,S., Gas,N.,
Petfalski,E., Tollervey,D., Mann,M., Hurt,E., Tschochner,H. et al.
(2001) Maturation and intranuclear transport of pre-ribosomes
requires Noc proteins. Cell, 105, 499–509.
33. de la Cruz,J., Sanz-Martinez,E. and Remacha,M. (2005) The
essential WD-repeat protein Rsa4p is required for rRNA processing
and intra-nuclear transport of 60S ribosomal subunits. Nucleic
Acids Res., 33, 5728–5739.
34. Hung,N.J., Lo,K.Y., Patel,S.S., Helmke,K. and Johnson,A.W.
(2008) Arx1 is a nuclear export receptor for the 60S ribosomal
subunit in yeast. Mol. Biol. Cell, 19, 735–744.
35. Bradatsch,B., Katahira,J., Kowalinski,E., Bange,G., Yao,W.,
Sekimoto,T., Baumgartel,V., Boese,G., Bassler,J., Wild,K. et al.
(2007) Arx1 functions as an unorthodox nuclear export receptor for
the 60S preribosomal subunit. Mol. Cell, 27, 767–779.
36. Lebreton,A., Saveanu,C., Decourty,L., Rain,J.C., Jacquier,A. and
Fromont-Racine,M. (2006) A functional network involved in the
recycling of nucleo-cytoplasmic pre-60S factors. J. Cell Biol., 173,
37. Pertschy,B., Saveanu,C., Zisser,G., Lebreton,A., Tengg,M.,
Jacquier,A., Liebminger,E., Nobis,B., Kappel,L., van der Klei,I.
et al. (2007) Cytoplasmic recycling of 60S preribosomal factors
depends on the AAA protein Drg1. Mol. Cell Biol., 27, 6581–6592.
38. De Marchis,M.L., Giorgi,A., Schinina,M.E., Bozzoni,I. and
Fatica,A. (2005) Rrp15p, a novel component of pre-ribosomal
particles required for 60S ribosome subunit maturation. RNA, 11,
39. Berges,T., Petfalski,E., Tollervey,D. and Hurt,E.C. (1994) Synthetic
lethality with fibrillarin identifies NOP77p, a nucleolar protein
required for pre-rRNA processing and modification. EMBO J., 13,
40. Daugeron,M.C., Kressler,D. and Linder,P. (2001) Dbp9p, a puta-
tive ATP-dependent RNA helicase involved in 60S-ribosomal-sub-
unit biogenesis, functionally interacts with Dbp6p. RNA, 7,
41. Daugeron,M.C. and Linder,P. (1998) Dbp7p, a putative ATP-
dependent RNA helicase from Saccharomyces cerevisiae, is required
for 60S ribosomal subunit assembly. RNA, 4, 566–581.
42. Horsey,E.W., Jakovljevic,J., Miles,T.D., Harnpicharnchai,P. and
Woolford,J.L. Jr. (2004) Role of the yeast Rrp1 protein in the
dynamics of pre-ribosome maturation. RNA, 10, 813–827.
Nucleic Acids Research, 2008, Vol. 36, No. 15 4999